Interaction of hnRNPA1/A2 and DAZAP1 with an Alu-Derived Intronic Splicing Enhancer Regulates ATM Aberrant Splicing

We have previously identified an Alu-derived Intronic Splicing enhancer (ISE) in the Ataxia Teleangectasia Mutated gene (ATM) that facilitates intron pre-mRNA processing and leads to the inclusion of a cryptic exon in the final mRNA transcript. By using an RNA pull-down assay, we show here that hnRNPA1/A2, HuR and DAZAP1 splicing factors and DHX36 RNA helicase bind to the ISE. By functional studies (overexpression and siRNA experiments), we demonstrate that hnRNPA1 and DAZAP1 are indeed involved in ISE-dependent ATM cryptic exon activation, with hnRNPA1 acting negatively and DAZAP1 positively on splicing selection. On the contrary, HuR and DHX36 have no effect on ATM splicing pattern. These data suggest that splicing factors with both negative and positive effect can assemble on the intronic Alu repeats and regulate pre-mRNA splicing

Dephosphorylated NSSR1 Is Induced by Androgen in Mouse Epididymis and Phosphorylated NSSR1 Is Increased during Sperm Maturation

NSSR1 (Neural salient serine/arginine rich protein 1, alternatively SRp38) is a newly identified RNA splicing factor and predominantly expressed in neural tissues. Here, by Western blot analysis and immunofluorescent staining, we showed that the expression of dephosphorylated NSSR1 increased significantly during development of the caput epididymis. In adult mice, phosphorylated NSSR1 was mainly expressed in the apical side of epithelial cells, and dephosphorylated NSSR1 in caput epididymis was upregulated in a testosterone dependent manner. In addition, subcellular immunoreactive distribution of NSSR1 varied in different regions of the epididymis. With respect to the sperm, phosphorylated NSSR1 was detected in the mid-piece of the tail as well as the acrosome. Furthermore, NSSR1 was released from the sperm head during the capacitation and acrosome reaction. These findings for the first time provide the evidence for the potential roles of NSSR1 in sperm maturation and fertilization

Results of a randomized, double blind, placebo controlled, crossover trial of melatonin for treatment of Nocturia in adults with multiple sclerosis (MeNiMS)

© 2018 The Author(s). Background: Nocturia is a common urinary symptom of multiple sclerosis (MS) which can affect quality of life (QoL) adversely. Melatonin is a hormone known to regulate circadian rhythm and reduce smooth muscle activity such as in the bladder. There is limited evidence supporting use of melatonin to alleviate urinary frequency at night in the treatment of nocturia. The aim of this study was to evaluate the effect of melatonin on the mean number of nocturia episodes per night in patients with MS. Methods: A randomized, double blind, placebo controlled crossover trial was conducted. 34 patients with nocturia secondary to multiple sclerosis underwent a 4-day pre-treatment monitoring phase. The patients were randomized to receive either 2 mg per night (taken at bedtime) of capsulated sustained-release melatonin (Circadin®) or 1 placebo capsule for 6 weeks followed by a crossover to the other regimen for an additional 6 weeks after a 1-month washout period. Results: From the 26 patients who completed the study, there was no significant difference observed in the signs or symptoms of nocturia when taking 2 mg melatonin compared to placebo. The primary outcome measure, mean number of nocturia episodes on bladder diaries, was 1.8/night at baseline, and 1.4/night on melatonin, compared with 1.6 for placebo (Medians 1.70, 1.50, and 1.30 respectively, p = 0.85). There was also no significant difference seen in LUTS, QoL and sleep quality when taking melatonin. No significant safety concerns arose. Conclusions: This small study suggests that a low dose of melatonin taken at bedtime may be ineffective therapy for nocturia in MS. Trial registration: (EudraCT reference) 2012-00418321 registered: 25/01/13. ISRCTN Registry: ISRCTN38687869

Cross-Sample Validation Provides Enhanced Proteome Coverage in Rat Vocal Fold Mucosa

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high Mr ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high Mr glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories

A “Crossomics” Study Analysing Variability of Different Components in Peripheral Blood of Healthy Caucasoid Individuals

Background: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable. Methodology/Principal Findings: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex "crossomics'' analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4(+)T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset. Conclusions/Significance: Our study shows that the intra- individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective

p21WAF1/CIP1 Upregulation through the Stress Granule-Associated Protein CUGBP1 Confers Resistance to Bortezomib-Mediated Apoptosis

p21(WAF1/CIP1) is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown.We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib ( =  PS-341/Velcade). This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis.We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1

Pemphigus autoimmunity: Hypotheses and realities

The goal of contemporary research in pemphigus vulgaris and pemphigus foliaceus is to achieve and maintain clinical remission without corticosteroids. Recent advances of knowledge on pemphigus autoimmunity scrutinize old dogmas, resolve controversies, and open novel perspectives for treatment. Elucidation of intimate mechanisms of keratinocyte detachment and death in pemphigus has challenged the monopathogenic explanation of disease immunopathology. Over 50 organ-specific and non-organ-specific antigens can be targeted by pemphigus autoimmunity, including desmosomal cadherins and other adhesion molecules, PERP cholinergic and other cell membrane (CM) receptors, and mitochondrial proteins. The initial insult is sustained by the autoantibodies to the cell membrane receptor antigens triggering the intracellular signaling by Src, epidermal growth factor receptor kinase, protein kinases A and C, phospholipase C, mTOR, p38 MAPK, JNK, other tyrosine kinases, and calmodulin that cause basal cell shrinkage and ripping desmosomes off the CM. Autoantibodies synergize with effectors of apoptotic and oncotic pathways, serine proteases, and inflammatory cytokines to overcome the natural resistance and activate the cell death program in keratinocytes. The process of keratinocyte shrinkage/detachment and death via apoptosis/oncosis has been termed apoptolysis to emphasize that it is triggered by the same signal effectors and mediated by the same cell death enzymes. The natural course of pemphigus has improved due to a substantial progress in developing of the steroid-sparing therapies combining the immunosuppressive and direct anti-acantholytic effects. Further elucidation of the molecular mechanisms mediating immune dysregulation and apoptolysis in pemphigus should improve our understanding of disease pathogenesis and facilitate development of steroid-free treatment of patients